Semen freezing allows worldwide commercialization of equine genetic. Despite improvement of techniques, semen of 20% of stallions remains unfreezable. Currently, post-thaw semen quality is only determined by progressive motility, but its definition is not standardised. Spermatozoa are highly differentiated cells and freezing lesions can occur on DNA, membrane, mitochondria or acrosome. Current research focuses on prediction of freezability, improvement of freezing extenders and prevention of Reactive Oxygen Species (ROS) effects. Under its O2 form, oxygen is inactive and oxidase or oxygenase enzymes are required to produce ROS. Two pathways of ROS production in semen are described: the intrinsic pathway reflecting ROS escaping from the spermatozoon mitochondria and the extrinsic pathway corresponding to ROS produced by inflammatory cells. ROS induce DNA fragmentation, lipid peroxidation, apoptosis and decreased motility of spermatozoa. Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after lysis. It is responsible for formation of hypochlorous acid, a strong oxidant, which could damage spermatozoa. However, MPO presence and effects have never been investigated in equine semen. The first aim was to assay presence of MPO in equine thawed semen and to determine a relation between MPO concentration and post-thaw semen parameters. 35 straws from different stallions were analyzed. Post-thaw spermatozoa and MPO concentrations, viability, morphology, progressive and total motility were determined. Our study showed that thawed semen contains large amounts of free MPO. High MPO concentration samples showed lower total and progressive motility. Higher proportion of acrosome reaction was observed in late examinations of the high MPO concentration group. As MPO was present in frozen semen and did interfere with its quality, timing and origin of its release was determined during the freezing process. Forty ejaculates were frozen with a classical procedure. MPO ELISA and MPO immunocytochemistries (ICC) were assayed in raw semen, centrifugation supernatant, and after cooling down to 4°C. Post-thaw MPO concentration and spermogram parameters were determined. MPO concentration increased after cooling and thawing when compared to fresh semen. As temperature decreased, MPO was higher in post-thaw poor quality samples. Non-sperm cells (NSC) showed various degrees of MPO-ICC, and were mostly epithelial cells with nuclear picnosis. Elastase, another neutrophil pro-inflammatory enzyme, was also assayed in post-thaw semen. In twenty ejaculates, NSC concentration was determined in fresh semen. Post-thaw motilities were determined by CASA; MPO and elastase concentrations were assayed by ELISA. Post-thaw elastase concentrations were low and there was no difference according to semen quality. NSC or MPO concentrations were not correlated to elastase concentration. NSC concentration was higher in unfreezable semen and correlated to post-thaw MPO concentration. To confirm MPO release by NSC during freezing, the effect of washing semen with density gradient centrifugation (DGC) was then assayed on NSC and MPO concentrations. NSC and MPO concentrations were assessed at each step and MPO localization was performed by ICC. DGC washing decreased NSC and MPO concentrations in post-thaw semen and NSC were mainly remaining in DGC supernatant. MPO concentration was correlated with NSC concentration in the upper layer of the DGC supernatant and in post-thaw semen. NSC were epithelial cells showing MPO-ICC staining. Fresh semen MPO concentration had no effect on fresh or post-thaw semen quality, while post-thaw semen concentrations were correlated with decreased motility. To understand these findings, concentration, activity and structure of MPO present in seminal plasma, sperm-rich pellet and post-thaw semen were assayed. Factor inducing MPO release was determined by adding or not glycerol in samples stored at 4°C or 20°C. Total MPO was high in seminal plasma and thawed semen and low in sperm-rich pellet. Active MPO was high in sperm-rich pellet and low in seminal plasma and post-thaw semen. MPO concentrations were correlated in post-thaw and in semen cooled at 4°C with or without glycerol. Active MPO in sperm-rich pellet and post-thaw progressive motility were highly negatively correlated. MPO present in fresh semen is mainly the native inactive enzyme subunit. To confirm our previous findings, effect of active MPO and fresh or frozen-thawed NSC was assayed on purified spermatozoa motility, mitochondrial potential, membrane and acrosome integrity. Highest MPO concentration tested (50ng/ml) decreased motility. However, highest MPO concentration did not affect mitochondrial potential, membrane or acrosome integrity. Thawed NSC did decrease motility and mitochondrial potential when compared to fresh NSC, suggesting a synergic effect between MPO and other products released by NSC after thawing. Temperature decrease during freezing process increases MPO concentration and post-thaw concentration is negatively associated to post-thaw motilities. ICC slides have shown MPO presence on epithelial keratinized and pycnotic cells while neutrophils were rarely observed. Semen washing with DGC decreases MPO and NSC concentrations in post-thaw semen as NSC and MPO concentrations were positively correlated. MPO present in seminal plasma is native and inactive form while MPO present in sperm-rich pellet is active and negatively correlated to the post-thaw progressive motility. Addition of active MPO in semen decreased motility but had no effect on acrosome integrity, despite what had previously been suggested. Thawed NSC addition to spermatozoa decreased mitochondrial potential, suggesting a synergic effect between MPO and other factors released by NSC. Further studies should investigate the origin of high inactive MPO concentrations in fresh semen. Other studies should be conduced about the origin of epithelial keratinized pyknotic NSC in fresh semen and the pathophysiological mechanism leading to their MPO release during freezing. Large scale studies should be conducted to confirm the use of NSC concentration in fresh semen or active MPO concentration in sperm rich pellet as freezability prognosis. Further studies should also investigate effect of MPO specific inhibitors. [less ▲]

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutro- phils during degranulation or after lysis. Post-thaw semen contains MPO, and concen- tration of this enzyme is associated ... [more ▼]

Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutro- phils during degranulation or after lysis. Post-thaw semen contains MPO, and concen- tration of this enzyme is associated with decreased motility. The aim of this study was to determine kinetics of MPO concentration during freezing, its origin, and its impact on frozenethawed semen. Forty ejaculates were used. Semen was frozen using the classical freezing procedure. MPO concentrations were assayed in fresh semen, after centrifuga- tion, and after cooling down to 4 C. Post-thaw MPO assay results and spermogram characteristics were determined. MPO immunocytochemistry was performed on 4 different ejaculates at each step of freezing procedure. MPO concentration increased after cooling down to 4 C and thawing compared with fresh semen. As temperature decreased, MPO was higher or tended to be higher in post-thaw poor quality samples. Nonsperm cells showed various degrees of MPO immunostaining and were observed as epithelial cells with nuclear pyknosis and keratinization. MPO immunostaining increased in medium and decreased in nonsperm cells during freezing. Our study shows that MPO concentration in equine semen increases when temperature decreases. We hypothesize that nonsperm cells present in fresh semen could release MPO. [less ▲]

In Nile tilapia (Oreochromis niloticus), individuals with atypical sexual genotype are commonly used in farming (use of YY males to produce all-male offsprings), but they also constitute major tools to ... [more ▼]

In Nile tilapia (Oreochromis niloticus), individuals with atypical sexual genotype are commonly used in farming (use of YY males to produce all-male offsprings), but they also constitute major tools to study sex determinism mechanisms. In other species, sexual genotype and sex reversal procedures affect different aspects of biology such as growth, behaviour and reproductive success. The aim of this study was to assess the influence of sexual genotype on sperm quality in Nile tilapia. Milt characteristics were compared in XX (sex-reversed), XY and YY males in terms of gonadosomatic index, sperm count, sperm motility and duration of sperm motility. Sperm motility was measured by computer-assisted sperm analysis (CASA) quantifying several parameters: total motility, progressive motility, curvilinear velocity, straight line velocity, average path velocity and linearity. None of the sperm trait measured differed significantly between the three genotypes. Mean values of gonadosomatic index, sperm concentration and sperm motility duration of XX, XY and YY males respectively ranged from 0.92 to 1.33 %, from 1.69 to 2.22 × 10(9) cells mL-1 and from 18’04’’ to 27’32’’. Mean values of total motility and curvilinear velocity 1 min after sperm activation respectively ranged from 53 to 58 % and from 71 to 76 µm s-1 for the three genotypes. After 3 min of activity, all the sperm motility and velocity parameters dropped by half and continued to slowly decrease thereafter. Seven min after activation, only 9 to 13 % of spermatozoa were still progressive. Our results prove that neither sexual genotype nor hormonal sex reversal treatments affect sperm quality in male Nile tilapias with atypical sexual genotype. [less ▲]

Aim of this paper is to review equine semen freezing procedures. Spermatozoa formation inside the testis, spermatozoa anatomy and physiology will be summarized, highlighting structures and/or functions injured during freezing/thawing process. Current quality standards will be defined for fresh and thawed semen with a special attention on total and progressive motilities and different definitions of these parameters in literature. New ways of semen analysis (as flow cytometry) will be discussed for their scientific and clinical implications. Freezing procedures will and new freezing procedures will be discussed. Future developments and progress in freezing methods will be exposed, as prediction of post-thaw semen quality on fresh semen basis, improvement of freezing extenders and prevention of reactive oxygen species effects. [less ▲]

Despite improvement of techniques, semen of 20% of stallions remains unfreezable. Recent studies focused on impact of reactive oxygen species and oxidant enzymes on semen characteristics. Myeloperoxidase (MPO) is a pro-oxidant enzyme contained in and released by neutrophils during degranulation or after cell lysis. It is responsible for the formation of hypochlorous acid, a strong oxidant agent which could damage spermatozoa. The aim of this study was to determine the relation between MPO concentration and characteristics of frozen semen from stallions. Thirty five straws from different stallions were analyzed. Post-thawing spermatozoal concentration, progressive and total motility were determined by CASA. Freezability was determined according to post-thawing progressive motility (over or under 15%). Percent of alive spermatozoa and abnormal forms were determined after Eosin-Nigrosin and Diff-Quick® staining respectively. Post-thawing MPO concentration was measured by ELISA. Our study shows that frozen thawed semen contains large amounts of free MPO. We also observed that post-thawing MPO ELISA assay can be used as an indicator of equine semen freezability. High MPO concentration samples showed lower total and progressive motility. A higher proportion of abnormal head shape associated with acrosome reaction was observed in our late examinations of the high concentration MPO group. Our results show that MPO adversely affects total and progressive motility of equine semen. A negative correlation between normal motile forms and MPO concentration was also observed. The effect of MPO on dead or abnormal forms remains to be precised. [less ▲]

Non sperm cells concentration is higher in unfreezable semen. A relation between non-sperm cells in fresh semen and MPO and post thawing quality has been observed. Neutrophil Elastase seems to have no ... [more ▼]

Non sperm cells concentration is higher in unfreezable semen. A relation between non-sperm cells in fresh semen and MPO and post thawing quality has been observed. Neutrophil Elastase seems to have no effect on semen characteristics and to be not associated with on-sperm cells and freezability. [less ▲]

In various species a relationship between oocyte maturity and morphological characteristics of oocytes has been established. To our knowledge, immature oocyte aspect under light microscopy has been paid ... [more ▼]

In various species a relationship between oocyte maturity and morphological characteristics of oocytes has been established. To our knowledge, immature oocyte aspect under light microscopy has been paid little attention in the equine. The aim of the study is to give a general description and analyse microscopic parameters of immature oocytes. Follicles from abattoir ovaries are punctured after measurement of their diameter and their cumulus-oocyte complexes are recovered. Different characters such as oocyte diameter, cumulus aspect, granulosity and polarity of ooplasm are observed under light microscopy. No correlation between estral activity, follicle maturity, cumulus cells aspect, polarity and granulosity of ooplasm was observed. Thickness of zona pellucida differs between oocytes with polar or non polar ooplasm. No correlation was observed between follicle diameter and oocyte diameter and none of the other studied parameters showed an influence on oocyte diameter. Results show that no character of oocytes observed under light microscopy can be related to follicular origin or to any other character. Further studies have to be done to compare these immature oocytes with mature oocytes and to find the ultra-structural origin of characters observed under light microscopy. [less ▲]

In various species a relationship between oocyte maturity and morphological characteristics of oocytes has been established. To our knowledge, little attention has been paid to the immature oocyte aspect ... [more ▼]

In various species a relationship between oocyte maturity and morphological characteristics of oocytes has been established. To our knowledge, little attention has been paid to the immature oocyte aspect under light microscopy in the equine. The aim of the study is to give a general description and analysis of the microscopic parameters of immature oocytes. Follicles from abattoir ovaries are punctured after measurement of their diameter and their cumulus-oocyte complexes are recovered. Diﬀerent characters such as oocyte diameter, cumulus aspect, granulosity and polarity of ooplasm are observed under light microscopy. No correlation between the estral activity, follicle maturity, cumulus cells aspect, polarity and granulosity of ooplasm was observed. The thickness of zona pellucida diﬀers between oocytes with polar or nonpolar ooplasm. No correlation was observed between the follicle and oocyte diameter and none of the other studied parameters showed an inﬂuence on the oocyte diameter. Results show that no character of the observed oocytes under light microscopy can be related to the follicular origin or to any other character. Further studies have to be done to compare these immature oocytes with mature ones and to ﬁnd the ultrastructural origin of the characters observed under light microscopy. [less ▲]